Cotton cultivar PHY 800 Pima

ABSTRACT

A cotton cultivar, designated PHY 800 Pima, is disclosed. The invention relates to the seeds of cotton cultivar PHY 800 Pima, to the plants and plant parts of cotton cultivar PHY 800 Pima and to methods for producing a cotton plant produced by crossing the cultivar PHY 800 Pima with itself or another cotton variety. The invention further relates to hybrid cotton seeds and plants produced by crossing the cultivar PHY 800 Pima with another cotton cultivar.

This application claims the benefit of U.S. Provisional Application No.60/530,157, filed Dec. 17, 2003.

BACKGROUND OF THE INVENTION

The present invention relates to a cotton (Gossypium) seed, a cottonplant, a part of a cotton plant, a cotton variety and a cotton hybrid.This invention further relates to a method for producing cotton seed andplants.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm. Incotton, the important traits include higher fiber (lint) yield, earliermaturity, improved fiber quality, resistance to diseases and insects,resistance to drought and heat, and improved agronomic traits.

Pureline cultivars of cotton are commonly bred by hybridization of twoor more parents followed by selection. The complexity of inheritance,the breeding objectives and the available resources influence thebreeding method. Pedigree breeding, recurrent selection breeding andbackcross breeding are breeding methods commonly used in self-pollinatedcrops such as cotton. These methods refer to the manner in whichbreeding pools or populations are made in order to combine desirabletraits from two or more cultivars or various broad-based sources. Theprocedures commonly used for selection of desirable individuals orpopulations of individuals are called mass selection, plant-to-rowselection and single seed descent or modified single seed descent. One,or a combination of these selection methods, can be used in thedevelopment of a cultivar from a breeding population.

Pedigree breeding is primarily used to combine favorable genes into atotally new cultivar that is different in many traits than either parentused in the original cross. It is commonly used for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁ (filial generation 1)population. An F₂ population is produced by selfing F₁ plants. Selectionof desirable individual plants may begin as early as the F₂ generationwherein maximum gene segregation occurs. Individual plant selection canoccur for one or more generations. Successively, seed from each selectedplant can be planted in individual, identified rows or hills, known asprogeny rows or progeny hills, to evaluate the line and to increase theseed quantity or to further select individual plants. Once a progeny rowor progeny hill is selected as having desirable traits it becomes whatis known as a breeding line that is specifically identifiable from otherbreeding lines that were derived from the same original population. Atan advanced generation (i.e., F₅ or higher) seed of individual lines areevaluated in replicated testing. The best lines or a mixture ofphenotypically similar lines from the same original cross are tested forpotential release as new cultivars.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep, et al. 1979; Fehr,1987).

The single seed descent procedure in the strict sense refers to plantinga segregating population, harvesting one seed from every plant, andcombining these seeds into a bulk which is planted the next generation.When the population has been advanced to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The primary advantages of the seed descentprocedures are to delay selection until a high level of homozygosity(e.g., lack of gene segregation) is achieved in individual plants, andto move through these early generations quickly, usually through usingwinter nurseries.

The modified single seed descent procedure involves harvesting multipleseed (i.e., a single lock or a simple boll) from each plant in apopulation and combining them to form a bulk. Part of the bulk is usedto plant the next generation and part is put in reserve. This procedurehas been used to save labor at harvest and to maintain adequate seedquantities of the population.

Selection for desirable traits can occur at any segregating generation(F₂ and above). Selection pressure is exerted on a population by growingthe population in an environment where the desired trait is maximallyexpressed and the individuals or lines possessing the trait can beidentified. For instance, selection can occur for disease resistancewhen the plants or lines are grown in natural or artificially-induceddisease environments. The breeder selects only those individuals havinglittle or no disease which are thus assumed to be resistant.

Promising advanced breeding lines are thoroughly tested and compared topopular cultivars in environments representative of the commercialtarget area(s) for three or more years. The best lines havingsuperiority over the popular cultivars are candidates to become newcommercial cultivars. Those lines still deficient in a few traits arediscarded or utilized as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, usually take from seven to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior because for most traits the true genotypic value ismasked by other confounding plant traits or environmental factors. Onemethod of identifying a superior plant is to observe its performancerelative to other experimental lines and widely grown standardcultivars. For many traits a single observation is inconclusive andreplicated observations over time and space are required to provide agood estimate of a line's genetic worth.

The goal of a commercial cotton breeding program is to develop new,unique and superior cotton cultivars. The breeder initially selects andcrosses two or more parental lines followed by generation advancementand selection, thus producing many new genetic combinations. The breedercan theoretically generate billions of different genetic combinationsvia this procedure. The breeder has no direct control over which geneticcombinations will arise. Therefore, two breeders will never develop thesame line having the same traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made during and at the end of the growing season. The lines whichare developed are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce, with any reasonable likelihood, thesame cultivar twice by using the exact same original parents and thesame selection techniques. This unpredictability results in theexpenditure of large amounts of research moneys to develop superior newcotton cultivars.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, grower, processor and consumer for specialadvertising, marketing and commercial production practices and newproduct utilization. The testing preceding the release of a new cultivarshould take into consideration research and development costs as well astechnical superiority of the final cultivar. For seed-propagatedcultivars, it must be feasible to produce seed easily and economically.

Cotton, Gossypium hirsutum (Acala) and Gossypium barbadense (Pima), areimportant and valuable field crops. Thus, a continuing goal of cottonplant breeders is to develop stable, high yielding cotton cultivars thatare agronomically sound. The reasons for this goal are obviously tomaximize the amount and quality of the fiber produced on the land usedand to supply fiber, oil and food for animals and humans. To accomplishthis goal, the cotton breeder must select and develop plants that havethe traits that result in superior cultivars.

The development of new cotton cultivars requires the evaluation andselection of parents and the crossing of these parents. The lack ofpredictable success of a given cross requires that a breeder, in anygiven year, make several crosses with the same or different breedingobjectives.

The cotton flower is monoecious in that the male and female structuresare in the same flower. The crossed or hybrid seed is produced by manualcrosses between selected parents. Floral buds of the parent that is tobe the female are emasculated prior to the opening of the flower bymanual removal of the male anthers. At flowering, the pollen fromflowers of the parent plants designated as male, are manually placed onthe stigma of the previously emasculated flower. Seed developed from thecross is known as first generation (F₁) hybrid seed. Planting of thisseed produces F₁ hybrid plants, of which half their genetic component isfrom the female parent and half from the male parent. Segregation ofgenes begins at meiosis thus producing second generation (F₂) seed.Assuming multiple genetic differences between the original parents, eachF₂ seed has a unique combination of genes.

SUMMARY OF THE INVENTION

The present invention relates to a cotton seed, a cotton plant, a partof a cotton plant, a cotton variety and a method for producing a cottonplant.

The present invention further relates to a method of producing cottonseeds and plants by crossing a plant of the instant invention withanother cotton plant.

This invention further relates to the seeds of cotton variety PHY 800Pima, to the plants of cotton variety PHY 800 Pima and to methods forproducing a cotton plant produced by crossing the cotton variety PHY 800Pima with itself or another cotton line. Thus, any such methods usingthe cotton variety PHY 800 Pima are part of this invention including:selfing, backcrosses, hybrid production, crosses to populations, and thelike.

In another aspect, the present invention provides for single traitconverted plants of PHY 800 Pima. The single transferred trait maypreferably be a dominant or recessive allele. Preferably, the singletransferred trait will confer such traits as herbicide resistance,insect resistance, resistance for bacterial, fungal, or viral disease,male fertility, male sterility, enhanced fiber quality, and industrialusage. The single trait may be a naturally occurring cotton gene or atransgene introduced through genetic engineering techniques.

In another aspect, the present invention provides a method ofintroducing a desired trait into cotton cultivar PHY 800 Pima whereinthe method comprises crossing a PHY 800 Pima plant with a plant ofanother cotton cultivar that comprises a desired trait to produce Flprogeny plants, wherein the desired trait is selected from the groupconsisting of herbicide resistance, insect resistance, and resistance tobacterial disease, fungal disease or viral disease; selecting progenyplants that have the desired trait to produce selected progeny plants;crossing the selected progeny plants with the PHY 800 Pima plants toproduce backcross progeny plants; selecting for backcross progeny plantsthat have the desired trait and physiological and morphologicalcharacteristics of cotton cultivar PHY 800 Pima to produce selectedbackcross progeny plants; and repeating these steps to produce selectedfirst or higher backcross progeny plants that comprise the desired traitand all of the physiological and morphological characteristics of cottoncultivar PHY 800 Pima as determined at the 5% significance level whengrown in the same environmental conditions. Included in this aspect ofthe invention is the plant produced by the method wherein the plant hasthe desired trait and all of the physiological and morphologicalcharacteristics of cotton cultivar PHY 800 Pima as determined at the 5%significance level when grown in the same environmental conditions.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of cotton plant PHY 800 Pima. The tissue culturewill preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing cottonplant, and of regenerating plants having substantially the same genotypeas the foregoing cotton plant. Preferably, the regenerable cells in suchtissue cultures will be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, orstems. Still further, the present invention provides cotton plantsregenerated from the tissue cultures of the invention.

Definitions

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Essentially all the phvsiological and momhological characteristics. Aplant having “essentially all the physiological and morphologicalcharacteristics” means a plant having the physiological andmorphological characteristics, except for the characteristics derivedfrom the converted trait.

Fallout (Fo). “Fallout” refers to the rating of how much cotton hasfallen on the ground at harvest.

Fiber Elongation (E1). “Fiber elongation” is defined as the measure ofelasticity of a bundle of fibers as measured by HVI.

Fiber Length (Len). “Fiber length” is defined as the upper half meanlength of fiber as measured by the HVI system in hundredths of an inch.

Fiber Strength (T1). “Fiber strength” is defined as the force requiredto break a bundle of fibers as measured in grams per tex on the HVI.

Foliar damage index. “Foliar damage index” refers to a scale for ratingfoliar damage caused by Fusarium oxysporum. A rating of 1 indicates nodamage symptoms evident, while a rating of 5 indicates severe orextensive damage symptoms.

Fruitinq Nodes. “Fruiting nodes” is defined as the number of nodes onthe main stem from which arise branches which bear fruit or bolls.

Gin Turnout. “Gin turnout” is defined as a fraction of lint in amachine-harvested sample of seed cotton (lint, seed, and trash)expressed as a percentage.

High Volume Instruments (HVI). “High Volume Instruments” (HVI) isdefined as a measurement system for the classification of Upland andAmerican Pima cotton fiber.

Length Uniformity Ratio. “Length uniformity ratio” is defined as theratio between the mean length and the upper half mean length expressedas a percentage.

Lint/boll. “Lint/boll” is the weight of lint per boll.

Lint Index. “Lint index” refers to the weight of lint per 100 seed ingrams.

Lint Percent. “Lint percent” is defined as the lint (fiber) fraction ofseed cotton (lint and seed).

Lint Yield. “Lint yield” is defined as the measure of the quantity offiber produced on a given unit of land. It is presented below in pounds(lbs) of lint per acre.

Maturity Rating (Matur). “Maturity rating” is defined as a visual ratingnear harvest on the amount of opened bolls on the plant.

Micronaire. “Micronaire” is defined as a measure of the fineness of thefiber. Within a cotton cultivar, micronaire is also a measure ofmaturity. Micronaire differences are governed by changes in perimeter orin cell wall thickness, or by changes in both. Within a variety, cottonperimeter is fairly constant and maturity will cause a change inmicronaire. Consequently, micronaire has a high correlation withmaturity within a variety of cotton. Maturity is the degree ofdevelopment of cell wall thickness. Micronaire may not have a goodcorrelation with maturity between varieties of cotton having differentfiber perimeter. Micronaire values range from about 2.0 to 6.0: Below2.9 Very fine Possible small perimeter but mature (good fiber), or largeperimeter but immature (bad fiber). 2.9 to 3.7 Fine Various degrees ofmaturity and/or perimeter. 3.8 to 4.6 Average Average degree of maturityand/or perimeter. 4.7 to 5.5 Coarse Usually fully developed (mature),but larger perimeter. 5.6+ Very Fully developed, large-perimeter fiber.coarse

Plant Height. “Plant height” is defined as the average height in inchesof a group of plants.

Resistance. “Resistance” is defined as the ability of plants to restrictthe activities of a specified pest, such as an insect, fungus, virus, orbacterium.

Root vascular stain index. “Root vascular stain index” refers to a scalefor rating root vascular stain caused by Fusarium oxysporum. A rating of1 indicates no root vascular stain evident, while a rating of 5indicates severe or extensive root vascular stain.

Seed/boll. “Seed/boll” refers to the number of seeds per boll.

Seedcotton/boll. “Seedcotton/boll” refers to the weight of seedcottonper boll in grams.

Seed Index (SI). “Seed index” is defined as the weight of 100 fuzzyseeds in grams.

Seed Integrity. “Seed integrity” is defined as a visual rating of thebreakage of whole seed or seed coat fragments in a gin-processed fuzzyseed sample. The rating ranges from 0 which indicates no breakage to 3which indicates severe breakage.

Seedweight (Sdwt). “Seedweight” is the weight of a given processed seedsample.

Single Trait Converted (Conversion). Single trait converted (conversion)plant refers to plants which are developed by a plant breeding techniquecalled backcrossing or via genetic engineering wherein essentially all(>94%) of the desired morphological and physiological characteristics ofa variety are recovered in addition to the single trait transferred intothe variety via the backcrossing technique or via genetic engineering.

Stringout Rating (So). “Stringout rating” is defined as a visual ratingprior to harvest of the relative looseness of the seed cotton held inthe boll structure on the plant.

Tolerance. “Tolerance” is defined as the ability of plants to endure aspecified pest (such as an insect, fungus, virus or bacterium) or anadverse environmental condition and still perform and produce in spiteof this disorder.

Vegetative Nodes. “Vegetative nodes” is defined as the number of nodesfrom the cotyledonary node to the first fruiting branch on the main stemof the plant.

VR. “VR” is defined as the allele designation for the single dominantallele of the present invention which confers virus resistance. TABLE 1VARIETY DESCRIPTION INFORMATION Species: Gossypium barbadense L. PlantCharacteristics: Plant Height:  38.1 inches Boll Weight:   3.72 g SeedIndex (g/100 seeds):  13.4 Seed Integrity:   1.45 Yield & Fiber QualityData: Lint Yield (Lbs/acre): 1598 Lint Percent:  40.0 Gin Turn-out: 34.5 Micronaire:   3.81 Fiber length:   1.42 Uniformity ratio:  88.1Fiber strength T1 (g/Tex):  42.7 Fiber elongation E1:   5.7

Disease Reaction

Fusarium oxysporum vasinfectum (FOV): Resistant to Race 4

This invention is also directed to methods for producing a cotton plantby crossing a first parent cotton plant with a second parent cottonplant, wherein the first or second cotton plant is the cotton plant fromthe cultivar PHY 800 Pima. Further, both the first and second parentcotton plants may be the cultivar PHY 800 Pima (e.g., self-pollination).Therefore, any methods using the cultivar PHY 800 Pima are part of thisinvention: selfing, backcrosses, hybrid breeding, and crosses topopulations. Any plants produced using cultivar PHY 800 Pima as a parentare within the scope of this invention. As used herein, the term “plant”includes plant cells, plant protoplasts, plant cells of tissue culturefrom which cotton plants can be regenerated, plant calli, plant clumps,and plant cells that are intact in plants or parts of plants, such aspollen, flowers, embryos, ovules, seeds, pods, leaves, stems, roots,anthers and the like. Thus, another aspect of this invention is toprovide for cells which upon growth and differentiation produce acultivar having essentially all of the physiological and morphologicalcharacteristics of PHY 800 Pima.

Culture for expressing desired structural genes and cultured cells areknown in the art. Also as known in the art, cotton is transformable andregenerable such that whole plants containing and expressing desiredgenes under regulatory control may be obtained. General descriptions ofplant expression vectors and reporter genes and transformation protocolscan be found in Gruber, et al., “Vectors for Plant Transformation, inMethods in Plant Molecular Biology & Biotechnology” in Glich, et al.,(Eds. pp. 89-119, CRC Press, 1993). Moreover GUS expression vectors andGUS gene cassettes are available from Clone Tech Laboratories, Inc.,Palo Alto, Calif. while luciferase expression vectors and luciferasegene cassettes are available from Pro Mega Corp. (Madison, Wis.).General methods of culturing plant tissues are provided, for example, byMaki, et al., “Procedures for Introducing Foreign DNA into Plants” inMethods in Plant Molecular Biology & Biotechnology, Glich, et al., (Eds.pp. 67-88 CRC Press, 1993); and by Phillips, et al., “Cell-TissueCulture and In-Vitro Manipulation” in Corn & Corn Improvement, 3rdEdition; Sprague, et al., (Eds. pp. 345-387) American Society ofAgronomy Inc., 1988 and U. S. Pat. No. 5,244,802. Methods of introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant cells with Agrobacterium tumefaciens, Horsch etal., Science, 227:1229 (1985). Descriptions of Agrobacterium vectorssystems and methods for Agrobacterium-mediated gene transfer areprovided by Gruber, et al., supra.

Useful methods include but are not limited to expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably expression vectors are introduced into planttissues using microprojectile-mediated delivery with a biolistic deviceor using Agrobacterium-mediated transformation. Transformant plantsobtained with the protoplasm of the invention are intended to be withinthe scope of this invention.

The present invention contemplates a cotton plant regenerated from atissue culture of a variety (e.g., PHY 800 Pima) or hybrid plant of thepresent invention. As is well known in the art, tissue culture of cottoncan be used for the in vitro regeneration of a cotton plant. Tissueculture of various tissues of cotton and regeneration of plantstherefrom is well known and widely published.

When the term cotton plant is used in the context of the presentinvention, this also includes any single trait conversions of thatvariety. The term single trait converted plant as used herein refers tothose cotton plants which are developed by a plant breeding techniquecalled backcrossing or via genetic engineering wherein essentially allof the desired morphological and physiological characteristics of avariety are recovered in addition to the single trait transferred intothe variety. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the variety. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to the recurrent parent. The parental cotton plant whichcontributes the trait for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental cotton plant to which the traitor traits from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol (Poehlman & Sleper, 1994; Fehr, 1987). In a typical backcrossprotocol, the original variety of interest (recurrent parent) is crossedto a second variety (nonrecurrent parent) that carries the single geneof interest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a cotton plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a gene or genes of the recurrent varietyare modified or substituted with the desired gene(s) from thenonrecurrent parent while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original variety. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable and/or agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many traits have been identified that are not regularly selected for inthe development of a new variety but that can be improved bybackcrossing techniques. Traits may or may not be transgenic; examplesof these traits include, but are not limited to, cytoplasmic or nuclearmale sterility, herbicide resistance, resistance for bacterial, fungal,or viral disease, insect resistance, male fertility, enhanced fiberquality, industrial usage, yield stability and yield enhancement. Thesetraits are generally inherited through the nucleus.

FURTHER EMBODIMENTS OF THE INVENTION

With the advent of molecular biological techniques allowing theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention in particular embodiments also relates to transformed versionsof the claimed variety or line.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid and can be used alone or incombination with other plasmids to provide transformed cotton plantsusing transformation methods as described below to incorporatetransgenes into the genetic material of the cotton plant(s).

Expression Vectors for Cotton Transformation: Marker Genes—Expressionvectors include at least one genetic marker, operably linked to aregulatory element (a promoter, for example) that allows transformedcells containing the marker to be either recovered by negativeselection, i.e., inhibiting growth of cells that do not contain theselectable marker gene, or by positive selection, i.e., screening forthe product encoded by the genetic marker. Many commonly used selectablemarker genes for plant transformation are well known in thetransformation arts and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). Anothercommonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Hayford et al., PlantPhysiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet, 210:86 (1987),Svab et al., Plant Mol. Biol. 14:197 (1990) Hille et al., Plant Mol.Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988).

Selectable marker genes for plant transformation which are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shahet al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643(1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase and chloramphenicol acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available. Molecular Probes publication2908, Imagene Green™, p. 1-4(1993) and Naleway et al., J. Cell Biol.115:151a (1991). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

A gene encoding Green Fluorescent Protein (GFP) has been utilized as amarker for gene expression in prokaryotic and eukaryotic cells. Chalfieet al., Science 263:802 (1994). GFP and mutants of GFP may be used asscreenable markers.

Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in a certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions, the presence of light,interactions with chemical substances or contact with other organismsincluding, but not limited to, certain pathogens. Tissue-specific,tissue-preferred, cell type specific, and inducible promoters constitutethe class of “non-constitutive” promoters. A “constitutive” promoter isa promoter which is active under most environmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in cotton. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in cotton. With aninducible promoter the rate of transcription increases in response to aninducing agent.

Any inducible promoter operable in plants can be used in the instantinvention. See Ward et al., Plant Mol. Biol. 22:361-366 (1993).Exemplary inducible promoters include, but are not limited to, that fromthe ACEI system which responds to copper (Mett et al., PNAS 90:4567-4571(1993)); In2 gene from maize which responds to benzenesulfonamideherbicide safeners (Hershey et al., Mol. Gen Genetics 227:229-237 (1991)and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressorfrom Tn10 (Gatz et al., Mol. Gen. Genetics 227:229-237 (1991)). Aparticularly preferred inducible promoter is a promoter that responds toan inducing agent to which plants do not normally respond. An exemplaryinducible promoter is the inducible promoter from a steroid hormonegene, the transcriptional activity of which is induced by aglucocorticosteroid hormone (Schena et al., Proc. Natl. Acad. Sci.U.S.A. 88:0421 (1991)).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in cotton or the constitutive promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in cotton.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, Xba1/Ncol fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similar to said Xba1/Ncolfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530. The AHAS promoter is another useful promoteras described in Grula, et al., Plant Mol. Biol. 28:837-846 (1995).

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in cotton.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in cotton. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promotersuch as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al., PlantMol. Biol. 9:3-17 (1987); Lerner et al., Plant Physiol. 91:124-129(1989); Fontes et al., Plant Cell 3:483-496 (1991); Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991); Gould et al., J. Cell. Biol.108:1657 (1989); Creissen et al., Plant J. 2:129 (1991); Kalderon, etal, Cell 39:499-509 (1984); Steifel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes—With transgenic plantsaccording to the present invention, a foreign protein can be produced incommercial quantities. Thus, techniques for the selection andpropagation of transformed plants, which are well understood in the art,yield a plurality of transgenic plants which are harvested in aconventional manner, and a foreign protein can then be extracted from atissue of interest or from total biomass. Protein extraction from plantbiomass can be accomplished by known methods which are discussed, forexample, by Heney and Orr, Anal. Biochem. 114:92-6 (1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a cotton plant. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes That Confer Resistance to Pests or Disease and That Encode:

-   -   A. Plant disease resistance genes. Plant defenses are often        activated by specific interaction between the product of a        disease resistance gene (R) in the plant and the product of a        corresponding avirulence (Avr) gene in the pathogen. A plant        variety can be transformed with cloned resistance gene to        engineer plants that are resistant to specific pathogen strains.        See, for example Jones et al., Science 266:789 (1994) (cloning        of the tomato Cf-9 gene for resistance to Cladosporium fulvum);        Martin et al., Science 262:1432 (1993) (tomato Pto gene for        resistance to Pseudomonas syringae pv. tomato encodes a protein        kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2        gene for resistance to Pseudomonas syringae).    -   B. A gene conferring resistance to a pest, such as nematodes.        See e.g., PCT Applications WO96/30517 and WO93/19181.    -   C. A Bacillus thuringiensis protein, a derivative thereof or a        synthetic polypeptide modeled thereon. See, for example, Geiser        et al., Gene 48:109 (1986), who disclose the cloning and        nucleotide sequence of a Bt δ-endotoxin gene. Moreover, DNA        molecules encoding δ-endotoxin genes can be purchased from        American Type Culture Collection, Manassas, Virginia, for        example, under ATCC Accession Nos. 40098, 67136, 31995 and        31998.    -   D. A lectin. See, for example, the disclosure by Van Damme et        al., Plant Molec. Biol. 24:25 (1994), who disclose the        nucleotide sequences of several Clivia miniata mannose-binding        lectin genes.    -   E. A vitamin-binding protein such as avidin. See PCT application        U.S. Ser. No. 93/06487. The application teaches the use of        avidin and avidin homologues as larvicides against insect pests.    -   F. An enzyme inhibitor, for example, a protease or proteinase        inhibitor or an amylase inhibitor. See, for example, Abe et        al., J. Biol. Chem. 262:16793 (1987) (nucleotide sequence of        rice cysteine proteinase inhibitor), Huub et al., Plant Molec.        Biol. 21:985 (1993) (nucleotide sequence of cDNA encoding        tobacco proteinase inhibitor 1), Sumitani et al., Biosci.        Biotech. Biochem. 57:1243 (1993) (nucleotide sequence of        Streptomyces nitrosporeus α-amylase inhibitor) and U.S. Pat. No.        5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).    -   G. An insect-specific hormone or pheromone such as an        ecdysteroid or juvenile hormone, a variant thereof, a mimetic        based thereon, or an antagonist or agonist thereof. See, for        example, the disclosure by Hammock et al., Nature 344:458        (1990), of baculovirus expression of cloned juvenile hormone        esterase, an inactivator of juvenile hormone.    -   H. An insect-specific peptide or neuropeptide which, upon        expression, disrupts the physiology of the affected pest. For        example, see the disclosures of Regan, J. Biol. Chem.        269:9 (1994) (expression cloning yields DNA coding for insect        diuretic hormone receptor), and Pratt et al., Biochem. Biophys.        Res. Comm. 163:1243 (1989) (an allostatin is identified in        Diploptera puntata). See also U.S. Pat. No. 5,266,317 to        Tomalski et al., who disclose genes encoding insect-specific,        paralytic neurotoxins.    -   I. An insect-specific venom produced in nature by a snake, a        wasp, etc. For example, see Pang et al., Gene 116:165 (1992),        for disclosure of heterologous expression in plants of a gene        coding for a scorpion insectotoxic peptide.    -   J. An enzyme responsible for hyper-accumulation of a        monoterpene, a sesquiterpene, a steroid, a hydroxamic acid, a        phenylpropanoid derivative or another non-protein molecule with        insecticidal activity.    -   K. An enzyme involved in the modification, including the        post-translational modification of a biologically active        molecule; for example, a glycolytic enzyme, a proteolytic        enzyme, a lipolytic enzyme, a nuclease, a cyclase, a        transaminase, an esterase, a hydrolase, a phosphatase, a kinase,        a phosphorylase, a polymerase, an elastase, a chitinase and a        glucanase, whether natural or synthetic. See PCT application WO        93/02197 in the name of Scott et al., which discloses the        nucleotide sequence of a callase gene. DNA molecules which        contain chitinase-encoding sequences can be obtained, for        example, from the ATCC under Accession Nos. 39637 and 67152. See        also Kramer et al., Insect Biochem. Molec. Biol. 23:691 (1993),        who teach the nucleotide sequence of a cDNA encoding tobacco        hornworm chitinase, and Kawalleck et al., Plant Molec. Biol.        21:673 (1993), who provide the nucleotide sequence of the        parsley ubi4-2 polyubiquitin gene.    -   L. A molecule that stimulates signal transduction. For example,        see the disclosure by Botella et al., Plant Molec. Biol. 24:757        (1994), of nucleotide sequences for mung bean calmodulin cDNA        clones, and Griess et al., Plant Physiol. 104:1467 (1994), who        provide the nucleotide sequence of a maize calmodulin cDNA        clone.

M. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

N. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

O. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

P. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

-   -   Q. A virus-specific antibody. See, for example, Tavladoraki et        al., Nature 366:469 (1993), who show that transgenic plants        expressing recombinant antibody genes are protected from virus        attack.    -   R. A developmental-arrestive protein produced in nature by a        pathogen or a parasite. Thus, fungal        endo-α-1,4-D-polygalacturonases facilitate fungal colonization        and plant nutrient release by solubilizing plant cell wall        homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology        10:1436 (1992). The cloning and characterization of a gene which        encodes a bean endopolygalacturonase-inhibiting protein is        described by Toubart et al., Plant J. 2:367 (1992).    -   S. A developmental-arrestive protein produced in nature by a        plant. For example, Logemann et al., Bioi/Technology 10:305        (1992), have shown that transgenic plants expressing the barley        ribosome-inactivating gene have an increased resistance to        fungal disease.

2. Genes That Confer Resistance to an Herbicide, For Example:

-   -   A. An herbicide that inhibits the growing point or meristem,        such as an imidazolinone or a sulfonylurea. Exemplary genes in        this category code for mutant ALS and AHAS enzyme as described,        for example, by Lee et al., EMBO J. 7:1241 (1988), and Miki et        al., Theor. Appl. Genet. 80:449 (1990), respectively.    -   B. Glyphosate (resistance conferred by mutant        5-enolpyruvyl-shikimate-3-phosphate synthase (EPSP) and aroA        genes, respectively) and other phosphono compounds such as        glufosinate (phosphinothricin acetyl transferase, PAT and        Streptomyces hygroscopicus PAT, bar, genes), and pyridinoxy or        phenoxy proprionic acids and cyclohexones (ACCase        inhibitor-encoding genes). See, for example, U.S. Pat. No.        4,940,835 to Shah, et al., which discloses the nucleotide        sequence of a form of EPSP which can confer glyphosate        resistance. A DNA molecule encoding a mutant aroA gene can be        obtained under ATCC accession number 39256, and the nucleotide        sequence of the mutant gene is disclosed in U.S. Pat. No.        4,769,061 to Comai. European patent application No. 0 333 033 to        Kumada et al., and U. S. Pat. No. 4,975,374 to Goodman et al.,        disclose nucleotide sequences of glutamine synthetase genes        which confer resistance to herbicides such as        L-phosphinothricin. The nucleotide sequence of a PAT gene is        provided in European application No. 0 242 246 to Leemans et al.        DeGreef et al., Bio/Technology 7:61 (1989), describe the        production of transgenic plants that express chimeric bar genes        coding for PAT activity. Exemplary of genes conferring        resistance to phenoxy proprionic acids and cyclohexones such as        sethoxydim and haloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3        genes described by Marshall et al., Theor. Appl. Genet. 83:435        (1992).    -   C. An herbicide that inhibits photosynthesis, such as a triazine        (psbA and gs+ genes) or a benzonitrile (nitrilase gene).        Przibila et al., Plant Cell 3:169 (1991), describe the        transformation of Chlamydomonas with plasmids encoding mutant        psbA genes. Nucleotide sequences for nitrilase genes are        disclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA        molecules containing these genes are available under ATCC        Accession Nos. 53435, 67441, and 67442. Cloning and expression        of DNA coding for a glutathione S-transferase is described by        Hayes et al., Biochem. J. 285:173 (1992).

3. Genes That Confer or Contribute to a Value-Added Trait, Such as:

-   -   A. Modified fatty acid metabolism, for example, by transforming        a plant with an antisense gene of stearyl-ACP desaturase to        increase stearic acid content of the plant. See Knultzon et al.,        Proc. Natl. Acad. Sci. U.S.A. 89:2624 (1992).    -   B. Decreased phytate content—1) Introduction of a        phytase-encoding gene would enhance breakdown of phytate thus        adding more free phosphate to the transformed plant. For        example, see Van Hartingsveldt et al., Gene 127:87 (1993), for a        disclosure of the nucleotide sequence of an Aspergillus niger        phytase gene. 2) A gene could be introduced that reduced phytate        content. This could be accomplished by cloning and then        reintroducing DNA associated with the single allele which is        responsible for maize mutants characterized by low levels of        phytic acid. See Raboy et al., Maydica 35:383 (1990).    -   C. Modified carbohydrate composition effected, for example, by        transforming plants with a gene coding for an enzyme that alters        the branching pattern of starch. See Shiroza et al., J. Bacteol.        170:810 (1988) (nucleotide sequence of Streptococcus mutants        fructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet.        20:220 (1985) (nucleotide sequence of Bacillus subtilis        levansucrase gene), Pen et al., BiolTechnology 10:292 (1992)        (production of transgenic plants that express Bacillus        licheniformis α-amylase), Elliot et al., Plant Molec. Biol.        21:515 (1993) (nucleotide sequences of tomato invertase genes),        Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directed        mutagenesis of barley α-amylase gene), Fisher et al., Plant        Physiol. 102:1045 (1993) (maize endosperm starch branching        enzyme II), and Haigler et al., Proc. Beltwide Cotton Prod. Res.        Conf. p. 483 (2000) (transgenic cotton with improved fiber        micronaire, strength and length, and increased fiber weight).

Methods for Cotton Transformation—Numerous methods for planttransformation have been developed, including biological and physical,plant transformation protocols. See, for example, Miki et al.,“Procedures for Introducing Foreign DNA into Plants” in Methods in PlantMolecular Biology and Biotechnology, Glick B. R. and Thompson, J. E.Eds. (CRC Press, Inc., Boca Raton, 1993) pages 67-88. In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation” in Methods inPlant Molecular Biology and Biotechnology, Glick B. R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

-   -   A. Agrobacterium-mediated Transformation—One method for        introducing an expression vector into plants is based on the        natural transformation system of Agrobacterium. See, for        example, Horsch et al., Science 227:1229 (1985). A. tumefaciens        and A. rhizogenes are plant pathogenic soil bacteria which        genetically transform plant cells. The Ti and Ri plasmids of A.        tumefaciens and A. rhizogenes, respectively, carry genes        responsible for genetic transformation of the plant. See, for        example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).        Descriptions of Agrobacterium vector systems and methods for        Agrobacterium-mediated gene transfer are provided by Gruber et        al., supra, Miki et al., supra, and Moloney et al., Plant Cell        Reports 8:238 (1989). See also, U.S. Pat. No. 5,563,055        (Townsend and Thomas), issued Oct. 8, 1996.    -   B. Direct Gene Transfer—Several methods of plant transformation,        collectively referred to as direct gene transfer, have been        developed as an alternative to Agrobacterium-mediated        transformation. A generally applicable method of plant        transformation is microprojectile-mediated transformation        wherein DNA is carried on the surface of microprojectiles        measuring 1 to 4 μm. The expression vector is introduced into        plant tissues with a biolistic device that accelerates the        microprojectiles to speeds of 300 to 600 m/s which is sufficient        to penetrate plant cell walls and membranes. Sanford et al.,        Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech.        6:299 (1988), Klein et al., Bio/Technology 6:559-563 (1988),        Sanford, J. C., Physiol Plant 7:206 (1990), Klein et al.,        Biotechnology 10:268 (1992). See also U.S. Pat. No. 5,015,580        (Christou, et al.), issued May 14, 1991; U.S. Pat. No. 5,322,783        (Tomes, et al), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of Vllth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

Following transformation of cotton target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed with another (non-transformed or transformed) variety in orderto produce a new transgenic variety. Alternatively, a genetic traitwhich has been engineered into a particular cotton line using theforegoing transformation techniques could be moved into another lineusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite variety into anelite variety, or from a variety containing a foreign gene in its genomeinto a variety or varieties which do not contain that gene. As usedherein, “crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

Single Gene Conversion—When the term cotton plant is used in the contextof the present invention, this also includes any single gene conversionsof that variety. The term single gene converted plant as used hereinrefers to those cotton plants which are developed by a plant breedingtechnique called backcrossing wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to the single or relatively small number ofdesirable genes transferred into the variety via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the variety. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to the recurrent parent, i.e., backcrossing 2, 3, 4, 5, 6,7 or more times to the recurrent parent. The parental cotton plant whichcontributes the gene for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental cotton plant to which the gene orgenes from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol (Poehiman & Sleper, 1994; Fehr, 1987). In a typical backcrossprotocol, the original variety of interest (recurrent parent) is crossedto a second variety (nonrecurrent parent) that carries the single geneof interest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a cotton plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocoltypically is to alter or substitute a single trait or characteristic inthe original variety although more complex transfers are often designedand carried out. To accomplish this, a single gene of the recurrentvariety is modified or substituted with the desired gene from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original variety. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable and/or agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic; examples of these traits include, but are not limited to,male sterility, herbicide resistance, resistance for bacterial, fungal,or viral disease, insect resistance, male fertility, enhancednutritional quality, industrial usage, yield stability and yieldenhancement. These genes are generally inherited through the nucleus.Several of these single gene traits are described in U.S. Pat. Nos.5,959,185, 5,973,234 and 5,977,445, the disclosures of which arespecifically hereby incorporated by reference.

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of cotton andregeneration of plants therefrom is well known and widely published asdescribed in U.S. Pat. Nos. 5,244,802; 5,583,036; 5,834,292; 5,859,321;5,874,662; 6,040,504; 6,573,437; 6,620,990; 6,624,344 and 6,660,914 thedisclosures of which are specifically hereby incorporated by reference.For example, reference may be had to Komatsuda, T. et al., “Genotype XSucrose Interactions for Somatic Embryogenesis in Soybean,” Crop Sci.31:333-337 (1991); Stephens, P.A., et al., “Agronomic Evaluation ofTissue-Culture-Derived Soybean Plants,” Theor. Appl. Genet. (1991)82:633-635; Komatsuda, T. et al., “Maturation and Germination of SomaticEmbryos as Affected by Sucrose and Plant Growth Regulators in SoybeansGlycine gracilis Skvortz and Glycine max (L.) Merr.” Plant Cell, Tissueand Organ Culture, 28:103-113 (1992); Dhir, S. et al., “Regeneration ofFertile Plants from Protoplasts of Soybean (Glycine max L. Merr.);Genotypic Differences in Culture Response,” Plant Cell Reports (1992)11:285-289; Pandey, P. et al., “Plant Regeneration from Leaf andHypocotyl Explants of Glycine-wightii (W. and A.) VERDC. var.longicauda,” Japan J. Breed. 42:1-5 (1992); and Shetty, K., et al.,“Stimulation of In Vitro Shoot Organogenesis in Glycine max (Merrill.)by Allantoin and Amides,” Plant Science 81:245-251 (1992); as well as U.S. Pat. No. 5,024,944 issued Jun. 18,1991 to Collins et al., and U.S.Pat. No. 5,008,200 issued Apr. 16, 1991 to Ranch et al., the disclosuresof which are hereby incorporated herein in their entirety by reference.Thus, another aspect of this invention is to provide cells which upongrowth and differentiation produce cotton plants having thephysiological and morphological characteristics of cotton variety PHY800 Pima.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, pods, leaves,stems, roots, root tips, anthers, and the like. Means for preparing andmaintaining plant tissue culture are well known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and 5,977,445,described certain techniques, the disclosures of which are incorporatedherein by reference.

This invention also is directed to methods for producing a cotton plantby crossing a first parent cotton plant with a second parent cottonplant wherein the first or second parent cotton plant is a cotton plantof the variety PHY 800 Pima. Further, both first and second parentcotton plants can come from the cotton variety PHY 800 Pima. Thus, anysuch methods using the cotton variety PHY 800 Pima are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using cotton variety PHY800 Pima as a parent are within the scope of this invention, includingthose developed from varieties derived from cotton variety PHY 800 Pima.Advantageously, the cotton variety could be used in crosses with other,different, cotton plants to produce first generation (F₁) cotton hybridseeds and plants with superior characteristics. The variety of theinvention can also be used for transformation where exogenous genes areintroduced and expressed by the variety of the invention. Geneticvariants created either through traditional breeding methods usingvariety PHY 800 Pima or through transformation of PHY 800 Pima by any ofa number of protocols known to those of skill in the art are intended tobe within the scope of this invention.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which cotton plants can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, pods, leaves,roots, root tips, anthers, and the like.

Tables

As shown in Table 2 below, PHY 800 Pima is compared to cotton varietiesPHY 76 and Pima S-7. The asterisk (*) indicates a significant differencefrom PHY 800 Pima at the 5% level. TABLE 2 Trait PHY 800 Pima PHY 76Pima S-7 LSD .05 CV % 2002 Lint Yield (lbs/acre) Buena Vista 1606 1443* 957* 141 6.1 Corcoran El Rico 1134  874*  618* 157 10.4 Stratford 19201838 1711* 204 6.5 Dos Palos 1452 1714* 1216* 179 7.1 Mean 1528 14671126* 255 7.2 2001 Lint Yield (lbs/acre) Buena Vista 1684 1599* 1195* 853.3 Corcoran Stev. 28 2188 2120 1516* 241 7.2 Corcoran Goldberg 13891359 1178 322 14.2 Stratford 1410 1367 1172* 64 2.82 Mean 1668 16111266* 210 7.93 Boll Wt. (g) 3.72   3.44*   3.41* 0.10 4.4 Seed Index(g/100 seeds) 13.4  13.1  12.2* 0.3 3.5 Seed Integrity 1.45   1.35  1.65 0.41 42.3 Lint Percent 40.0  39.8  40.3 0.3 1.0 Gin Turn-out 34.5 33.6*  34.2 0.4 2.3

As shown in Table 3 below, the foliar damage index and the root vascularstain index for PHY 800 Pima is compared with cotton cultivars P.629 andDP744. TABLE 3 Index PHY 800 Pima P.629 DP744 Foliar damage indexLocation 1 0.1 2.8 3.1 Location 2 0.1 3.3 3.8 Root vascular stain indexLocation 1 0.1 4.2 4.7 Location 2 0.1 2.5 4.0

Deposit Information

A deposit of the Phytogen Seed Company, LLC proprietary cotton cultivarPHY 800 Pima disclosed above and recited in the appended claims has beenmade with the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110. The date of deposit was Dec. 2, 2003.The deposit of 2,500 seeds was taken from the same deposit maintained byPhytogen Seed Company, LLC since prior to the filing date of thisapplication. All restrictions upon the deposit have been removed, andthe deposit is intended to meet all of the requirements of 37 C.F.R.§1.801-1.809. The ATCC accession number is PTA-5667. The deposit will bemaintained in the depository for a period of 30 years, or 5 years afterthe last request, or for the effective life of the patent, whichever islonger, and will be replaced as necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

1. A seed of cotton cultivar designated PHY 800 Pima, representativeseed of said cultivar having been deposited under ATCC Accession No.PTA-5667.
 2. A cotton plant, or a part thereof, produced by growing theseed of claim
 1. 3. A tissue culture of regenerable cells produced fromthe plant of claim
 2. 4. Protoplasts produced from the tissue culture ofclaim
 3. 5. The tissue culture of claim 3, wherein cells of the tissueculture are from a plant part selected from the group consisting ofleaf, pollen, embryo, root, root tip, anther, pistil, flower, seed, bolland stem.
 6. A cotton plant regenerated from the tissue culture of claim3, said plant having all the morphological and physiologicalcharacteristics of cotton cultivar PHY 800 Pima, representative seed ofsaid cultivar having been deposited under ATCC Accession No. PTA-5667.7. A method for producing an F1 hybrid cotton seed, wherein the methodcomprises crossing the plant of claim 2 with a different cotton plantand harvesting the resultant Fl hybrid cotton seed.
 8. A hybrid cottonseed produced by the method of claim
 7. 9. A hybrid cotton plant, or apart thereof, produced by growing said hybrid seed of claim
 8. 10. Amethod of producing an herbicide resistant cotton plant wherein themethod comprises transforming the cotton plant of claim 2 with atransgene that confers herbicide resistance.
 11. An herbicide resistantcotton plant produced by the method of claim
 10. 12. The cotton plant ofclaim 11, wherein the transgene confers resistance to an herbicideselected from the group consisting of imidazolinone, sulfonylurea,glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.13. A method of producing an insect resistant cotton plant wherein themethod comprises transforming the cotton plant of claim 2 with atransgene that confers insect resistance.
 14. An insect resistant cottonplant produced by the method of claim
 13. 15. The cotton plant of claim14, wherein the transgene encodes a Bacillus thuringiensis endotoxin.16. A method of producing a disease resistant cotton plant wherein themethod comprises transforming the cotton plant of claim 2 with atransgene that confers disease resistance.
 17. A disease resistantcotton plant produced by the method of claim
 16. 18. A method ofintroducing a desired trait into cotton cultivar PHY 800 Pima whereinthe method comprises: (a) crossing PHY 800 Pima plants grown from PHY800 Pima seed, representative seed having been deposited under ATCCAccession No. PTA-5667, with plants of another cotton cultivar thatcomprise a desired trait to produce F1 progeny plants, wherein thedesired trait is selected from the group consisting of male sterility,herbicide resistance, insect resistance and disease resistance; (b)selecting F1 progeny plants that have the desired trait to produceselected Fl progeny plants; (c) crossing the selected progeny plantswith the PHY 800 Pima plants to produce backcross progeny plants; (d)selecting for backcross progeny plants that have the desired trait andphysiological and morphological characteristics of cotton cultivar PHY800 Pima to produce selected backcross progeny plants; and (e) repeatingsteps (c) and (d) three or more times in succession to produce selectedfourth or higher backcross progeny plants that comprise the desiredtrait and all of the physiological and morphological characteristics ofcotton cultivar PHY 800 Pima as shown in Table 1 and as determined atthe 5% significance level when grown in the same environmentalconditions.
 19. A plant produced by the method of claim 18, wherein theplant has the desired trait and all of the physiological andmorphological characteristics of cotton cultivar PHY 800 Pima as shownin Table 1 and as determined at the 5% significance level when grown inthe same environmental conditions.
 20. The plant of claim 19 wherein thedesired trait is herbicide resistance and the resistance is conferred toan herbicide selected from the group consisting of imidazolinone,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine andbenzonitrile.
 21. The plant of claim 19 wherein the desired trait isinsect resistance and the insect resistance is conferred by a transgeneencoding a Bacillus thuringiensis endotoxin.